Figure 5: Ep3 regulates CX3CR1 and VEGF expression through TGFβ1 signalling.
(a,b)Western blot analysis of CCR2, CX3CR1, phospho-Smad2, phospho-Smad3 and VEGF in Mos/Mps from Ep3F/F and Ep3F/F;LysMCre mice at 16 h (a) and 96 h (b)after a zymosan challenge. (c) Western blot analysis of CCR2, CX3CR1, phospho-Smad2, phospho-Smad3 and VEGF in cultured Mps with or without PGE2 stimulation. (d) Effect of Ep3 deletion on Mp migration in response to PGE2. Data represent mean±s.e.m. *P<0.05 versus Ep3F/F, #P<0.05 as indicated (unpaired two-tailed t-test); n=5. (e) Effect of TGFβ1 blocker SB525334 on Ep3-mediated VEGF and CX3CR1 mRNA expression in cultured Mps. Mp-Ep3Tg, Mp-specific Ep3α transgenic mice. Data represent mean±s.e.m. *P<0.05 versus wild-type (WT), **P<0.01 versus indicated (unpaired two-tailed t-test); n=5. (f) Representative immunostaining of CD68 (red) and VEGF (green) in peri-infarct zones of hearts from Mp-Ep3Tg mice at day 14 post MI. The solid box outlines the region enlarged below. Yellow arrow, CD68+/VEGF+ cells. Scale bar 20 μm. IZ, infarct zone. (g) Quantitation of VEGF signalling in CD68+ cells in injured hearts as shown in f. Data represent mean±s.e.m. *P<0.05 versus WT, #P<0.05 as indicated (unpaired two-tailed t-test); n=5. (h) Effect of SB525334 on Ep3-mediated VEGF and CX3CR1 protein expression in cultured Mps. (i) Effect of SB525334 on PGE2/Ep3-mediated Mp migration in vitro. Data represent mean±s.e.m. *P<0.05 versus WT, #P<0.05 as indicated (unpaired two-tailed t-test); n=4–6.
